Causatum of Probiotic Yeast Saccharomyces cerevisiae SBO1 Supplementation on Growth and Aflatoxin Amelioration in Broilers.

Probiotics are beneficial live microorganisms that benefit the host's health when administered in the required number. They play a vital role in preventing infectious diseases caused by pathogens. The current study aimed to discover a competent probiotic microbe that can detoxify aflatoxin and promote poultry health. The yeast isolate SBO1 tolerates the temperature of 42 °C, low pH, and high bile conditions, has good auto aggregation, hydrophobicity, and exhibits improved adherence to chick intestinal epithelial cells. In addition, it has an aflatoxin detoxifying ability of 56% after 24 h. In-vivo studies in broilers resulted in a higher body weight (2138 g) and greater feed conversion efficiency in the T2 group when fed with yeast SBO1-supplemented diet. Gizzard, spleen, and bursa Fabricius were all found to weigh the same, however, a significant difference (p < 0.05) was observed in the carcass, breast yield, and fat. Therefore it was determined that adding 0.2% yeast to the broiler diet increased performance by lessening the toxin's adverse effects. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01078-5.

Metagenomic Evidence of the Prevalence and Distribution Patterns of Antimicrobial Resistance Genes in Dairy Agroecosystems.

Antimicrobial resistance (AR) is a global problem with serious implications for public health. AR genes are frequently detected on animal farms, but little is known about their origin and distribution patterns. We hypothesized that AR genes can transfer from animal feces to the environment through manure, and to this end, we characterized and compared the resistomes (collections of AR genes) of animal feces, manure, and soil samples collected from five dairy farms using a metagenomics approach. Resistomes constituted only up to 1% of the total gene content, but were variable by sector and also farm. Broadly, the identified AR genes were associated with 18 antibiotic resistances classes across all samples; however, the most abundant genes were classified under multidrug transporters (44.75%), followed by resistance to vancomycin (12.48%), tetracycline (10.52%), bacitracin (10.43%), beta-lactam resistance (7.12%), and MLS efflux pump (6.86%) antimicrobials. The AR gene profiles were variable between farms. Farm 09 was categorized as a high risk farm, as a greater proportion of AR genes were common to at least three sectors, suggesting possible horizontal transfer of AR genes. Taxonomic characterization of AR genes revealed that a majority of AR genes were associated with the phylum Proteobacteria. Nonetheless, there were several members of Bacteroidetes, particularly Bacteroides genus and several lineages from Firmicutes that carried similar AR genes in different sectors, suggesting a strong potential for horizontal transfer of AR genes between unrelated bacterial hosts in different sectors of the farms. Further studies are required to affirm the horizontal gene transfer mechanisms between microbiomes of different sectors in animal agroecosystems.

Metagenomic assessment of the functional potential of the rumen microbiome in Holstein dairy cows.

The microbial ecology of the rumen microbiome is influenced by the diet and the physiological status of the dairy cow and can have tremendous influence on the yield and components of milk. There are significant differences in milk yields between first and subsequent lactations of dairy cows, but information on how the rumen microbiome changes as the dairy cow gets older has received little attention. We characterized the rumen microbiome of the dairy cow for phylogeny and functional pathways by lactation group and stage of lactation using a metagenomics approach. Our findings revealed that the rumen microbiome was dominated by Bacteroidetes (70%), Firmicutes (15-20%) and Proteobacteria (7%). The abundance of Firmicutes and Proteobacteria were independently influenced by diet and lactation. Bacteroidetes contributed to a majority of the metabolic functions in first lactation dairy cows while the contribution from Firmicutes and Proteobacteria increased incrementally in second and third lactation dairy cows. We found that nearly 70% of the CAZymes were oligosaccharide breaking enzymes which reflect the higher starch and fermentable sugars in the diet. The results of this study suggest that the rumen microbiome continues to evolve as the dairy cow advances in lactations and these changes may have a significant role in milk production.

In vitro assessment for the probiotic potential of Pichia kudriavzevii.

It is of interest to isolate the probiotic yeast Pichia kudriavzevii based on its probiotic characteristics and enzyme production. The isolate was able to withstand high acid, bile concentration and showed a high viability. Additionally, it showed auto aggregation ability that increases with time and hydrophobicity with xylene. It was resistant to different antibiotics and showed no hemolytic activity. The isolate was also capable of producing phytase that can break down phytate. Overall, the characteristics of P. kudriavzevii suggest that it could potentially have probiotic properties, and its ability to produce phytase could also make it useful in feed and animal industries.

Exploration of Antidiabetic, Cholesterol-Lowering, and Anticancer Upshot of Probiotic Bacterium Pediococcus pentosaceus OBK05 Strain of Buttermilk.

Chronic metabolic disorders such as hyperglycemia (diabetes), hyperlipidemia (high cholesterol), and cancer have become catastrophic diseases worldwide. Accordingly, probiotic intervention is a new approach for alleviating catastrophic diseases. In the present study, Pediococcus pentosaceus OBK05 was investigated as a potential probiotic bacterium for antidiabetic, cholesterol-lowering, and anticancer activities by in vitro and in vivo studies. Cell-free supernatant (CFS) of OBK05 showed potent antidiabetic activity by inhibition of α-amylase (72 ± 0.9%) and α-glucosidase (61 ± 0.8%) activity in vitro when compared to that of acarbose as standard (86 ± 0.9%). Furthermore, the in vivo cholesterol-lowering activity of OBK05 was studied using cholesterol-fed hypercholesterolemic mice. When compared to the cholesterol-fed group, the OBK05-intervened cholesterol-fed mice group had significantly lowered the levels of lipids and showed significantly lower lipid droplet accumulation in the histology of hepatocytes. Similarly, CFS exhibited higher anticancer activity (87.57 ± 1.27%) against HT-29 cells with an IC50 of 54.51 ± 1.8. Fluorescence-activated cell sorting (FACS) analysis revealed that CFS induced the cell cycle arrest by inhibiting the G1 to S phase transition. Further, in vivo anticancer activities were confirmed in BALB/c mice models, which were divided into four groups and held for 16 weeks. HT-29 cells (5×109/mice) were injected subcutaneously twice (0 and 2nd week), and OBK05 (3 ×105 CFU/mL) was given orally to the respective groups. The OBK05-intervened HT-29 cell-induced mice group showed significant improvement at the histological level and alleviated the degree of atypia in the intestine.

Antimycotic effect of 3-phenyllactic acid produced by probiotic bacterial isolates against Covid-19 associated mucormycosis causing fungi.

The Covid-19 associated mucormycosis (CAM) is an emerging disease affecting immunocompromised patients. Prevention of such infections using probiotics and their metabolites persist as effective therapeutic agents. Therefore, the present study emphasizes on assessment of their efficacy and safety. Samples from different sources like human milk, honey bee intestine, toddy, and dairy milk were collected, screened and characterized for potential probiotic lactic acid bacteria (LAB) and their metabolites to be used as effective antimicrobial agents to curtail CAM. Three isolates were selected based on probiotic properties and characterized as Lactobacillus pentosus BMOBR013, Lactobacillus pentosus BMOBR061 and Pediococcus acidilactici BMOBR041 by 16S rRNA sequencing and MALDI TOF-MS. The antimicrobial activity against standard bacterial pathogens showed ˃9 mm zone of inhibition. Furthermore, the antifungal activity of three isolates was tested against Aspergillus flavus MTCC 2788, Fusarium oxysporum, Candida albicans and Candida tropicalis where the results showed significant inhibition of each fungal strain. Further studies were carried out on lethal fungal pathogens like Rhizopus sp. and two Mucor sp. which are associated with post Covid-19 infection in immunosuppressed diabetic patients. Our studies on CAM inhibitory effect of LAB revealed the efficient inhibition against Rhizopus sp. and two Mucor sp. The cell free supernatants of three LAB showed varied inhibitory activity against these fungi. Following the antimicrobial activity, the antagonistic metabolite 3-Phenyllactic acid (PLA) in culture supernatant was quantified and characterized by HPLC and LC-MS using standard PLA (Sigma Aldrich). The isolate L. pentosus BMOBR013 produced highest PLA (0.441 g/L), followed by P. acidilactici BMOBR041 (0.294 g/L) and L. pentosus BMOBR061 (0.165 g/L). The minimum inhibitory concentration of HPLC eluted PLA on the Rhizopus sp. and two Mucor sp. was found to be 180 mg/ml which was further confirmed by inhibition of total mycelia under live cell imaging microscope.

Biotransformation of toxic lignin and aromatic compounds of lignocellulosic feedstock into eco-friendly biopolymers by Pseudomonas putida KT2440.

Lignin and its derivatives are the most neglected compounds in bio-processing industry due to their toxic and recalcitrant nature. Considering this, the present study aimed at valorizing these toxic compounds by employing Pseudomonas putida KT2440. Acclimatization resulted in improved tolerance with considerable lag phase reduction and aromatics degradation. Glucose as co-substrate enhanced growth and degradation in the toxic environment. The strain was able to degrade 30 % (1.60 g·L-1) lignin, 45 mM benzoate, 40 mM p-coumarate, 35 mM ferulate, 10 mM phenol, 10 mM pyrocatechol and 8 mM aromatics mixture. The strain synthesized biopolymers using these compounds under feast and famine conditions. Characterization using GC-MS, FT-IR, H1 NMR revealed them to be Polyhydroxyalkanoate (PHA) heteropolymers. All the analyzed PHAs contained versatile monomers with Hexadecanoic acid being the major one. This is a novel attempt towards lignin and aromatics degradation coupled with biopolymers synthesis without any genetic manipulation of the strain.

Unraveling the probiotic efficiency of bacterium Pediococcus pentosaceus OBK05 isolated from buttermilk: An in vitro study for cholesterol assimilation potential and antibiotic resistance status.

The present study describes the probiotic potential and functional properties of the lactic acid bacterium Pediococcus pentosaceus OBK05 isolated from buttermilk. The isolate OBK05 was assessed for its probiotic properties. The isolate showed notable tolerance to pH 2.0 and 3.0 (8.44, 8.35 log CFU/mL), oxbile of 0.5% at 2 and 4 h of incubation (6.97, 6.35 log CFU/mL) and higher aggregation (auto-aggregation, adhesion to hydrocarbons) than the referral strain, Lactobacillus acidophilus MTCC 10307. The adhesion efficiency to HT-29 cells was found to be maximum, corresponding to 93.5% and 97% at 1 and 2 h incubation, respectively. In addition, the isolate OBK05 showed antagonistic solid activity against bacterial pathogens like Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 1133. The phenotypic antibiotic resistance of the isolate was examined before and after curing plasmids. Among the known five structural genes responsible for different antibiotic resistance, four genes indicating antibiotic resistance to kanamycin-Aph (3´´)-III, streptomycin-strA, vancomycin-vanA and ciprofloxacin-gyrA were detected by PCR amplification of genomic DNA. Further, the horizontal gene transfer from OBK05 isolate to pathogens was not found for these antibiotic resistance markers when filter and food mating were carried out as no transconjugants developed on media plates containing respective antibiotics. This indicates that the intrinsic resistance is harbored on chromosomal genes, and hence it is nontransferable to other microbes. In addition, strain OBK05 exhibited good DPPH scavenging activity of 56 to 77% and liberated free amino acid from conjugated bile acid. The strain OBK05 demonstrated a strong ability to reduce cholesterol at 12 h (17%), 24 h (27%) and 48 h (67%) of incubation.

Probiotic Pediococcus acidilactici strain from tomato pickle displays anti-cancer activity and alleviates gut inflammation in-vitro.

The present study characterized the potential probiotic properties of Pediococcus acidilactici TMAB26 strain isolated from traditional Indian tomato pickle, and evaluated its possible therapeutic applications as an anti-cancer and anti-inflammatory agent in vitro. The 16S rRNA sequencing and primary screening demarcated TMAB26 strain as an ideal probiotic candidate, with distinctive properties of acid tolerance (58.02% at pH 2.5), bile tolerance (55.53% at 0.5%), and efficient adherence to the mucosal surface of the human intestinal cells in vitro, along with antagonistic, anti-inflammatory, and anti-cancer properties. The strain exhibited antagonism against standard intestinal pathogenic strains Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, E. coli, Klebsiella pneumonia, and Salmonella typhi with zones of inhibition in the range of 6-18 mm. The cytotoxicity evaluation of the probiotic isolate TMAB26 culture supernatant (1:1 dilution) showed significant cytotoxicity on HT-29 (94.91% ± 1.27) and Caco-2 (92.63% ± 0.63) cancer cells when compared to that of the peripheral blood mononuclear cells (PBMCs) alone. Furthermore, the strain culture supernatant reduced the mRNA levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) by threefold, Interleukin-6 (IL-6) by eightfold and increased the mRNA levels of the anti-inflammatory cytokine Interleukin-10 (IL-10) in lipopolysaccharide (LPS) pretreated HT-29 and peripheral blood mononuclear cells (PBMCs), suggesting the potential role of TMAB26 isolate, i.e., Pediococcus acidilactici MTCC 13014 in alleviating gut inflammation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02570-1.

Bioprospecting of Palmyra Palm (Borassus flabellifer) Nectar: Unveiling the Probiotic and Therapeutic Potential of the Traditional Rural Drink.

The present study investigates the therapeutic and probiotic attributes of traditional Toddy Palm Nectar (TPN). Glucose was found to be the highest with 4.37 mg/ml and arabinose was the least with 2.85 mg/ml. The average ethanol concentration of fresh TPN was found to be 0.3 mg/ml. The nutritional profile of TPN revealed 18 volatile fatty acids, the major one being hexadecenoic acid (M/Z 74). Amino acid profiling showed 26 amino acids, with OH-lysine-2 the highest (12.86%). About 120 morphologically distinct lactic acid bacteria (LAB) were isolated from 26 TPN samples, based on differential growth and in vitro probiotic characteristics. After 16S rRNA sequencing, four indigenous LAB strains were identified as Lactobacillus plantarum group OUBN1, Enterococcus faecium OUBN3, Pediococcus acidilactici OUBN4, and Pediococcus pentosaceous OUBN5 and their sequences were deposited to NCBI. Microbiological safety evaluation studies showed the absence of hemolytic, gelatinolytic and proteolytic activity. The bacterial isolate OUBN3 showed a maximum survival rate of 6.91 ± 0.04 log cfu/ml at acidic pH 2.5 and isolate OUBN5 showed 6.94 ± 0.02 log cfu/ml at pH 3.0. Similarly, the isolate OUBN5 showed 7.92 ± 0.03 log cfu/ml to 0.3% ox-bile after 4 h and 8.94 ± 0.03 log cfu/ml to simulated gastric juice after 3 h of treatments. OUBN1 expressed the highest autoaggregation (81.76 ± 1.25%), cell surface hydrophobicity (79.71 ± 3.42%), and displayed the maximum coaggregation with E. coli MTCC452 (76.96%), K. pneumoniae MTCC109 (75.62%), and S. aureus MTCC902 (70.69%). All strains showed significant antibiotic and antimicrobial activity. Isolate OUBN1 displayed hydroxyl radical scavenging activity (68.71 ± 1.0%) with an IC50 value of 75.62 µg/ml and the highest anti-cancer activity (percentage inhibition of 88.55) against HT-29 cells. Based on the characteristics observed, L. plantarum group OUBN1 and P. pentosaceous OUBN5 were found to be potential isolates to employ as probiotic microbiota in food and forage preparations. These findings reinforce the fact that LAB isolated from TPN could be exploited as an alternative means toward potential therapeutic applications.

Optimized Production of Xylanase by Penicillium purpurogenum and Ultrasound Impact on Enzyme Kinetics for the Production of Monomeric Sugars From Pretreated Corn Cobs.

Corn cob is an abundant organic source with significant potential in sustainable energy development. For the effective conversion of the feedstocks to valued commodities, effective biocatalysts are highly desired. The present study aims at optimizing the critical parameters required for xylanase production by Penicillium purpurogenum isolated from rotten wood sample using the Taguchi orthogonal array layout of L25 (5∧6). The optimized conditions like temperature 40°C, pH 3, size of inoculum 1.2 × 108 spores/ml, moisture 70%, peptone 0.8%, and 5 days of incubation resulted in 1,097 ± 6.76 U/gram dry substrate (gds) xylanase which was 65.72% more when compared to un-optimized production of xylanase. The xylanase thus produced, effectively carried out pretreated corn cob saccharification and the reaction was further improved with ultrasound assistance which has increased the saccharification yield to 12.02% along with significant reduction in reaction time. The saccharification efficiency of pretreated corn cob was found to be 80.29% more compared to the raw corn cob, reflecting its recalcitrance to digestion. Indeed, xylan being the second most abundant polymer in lignocellulosic biomass, considerable attention is being paid for its effective conversion to valued products.

Cloning and in vivo metabolizing activity study of CYP3A4 on amiodarone drug residues: A possible probiotic and therapeutic option.

BACKGROUND: Amiodarone represents a principal antiarrhythmic pharmaceutical drug available in the market for the treatment of ventricular arrhythmias. However, despite its better efficacy, the usage of amiodarone is associated with extracardiac toxicity. The human body evolved a system of cytochrome P450 enzymes which play an essential role in the metabolism of harmful foreign substances. Therefore, CYP enzymes may either lead to the elimination or degradation of the leftover drug residues. OBJECTIVE: The present study focused on successful utilization of Saccharomyces cerevisiae strain OBS2 with probiotic- cum- therapeutic potential and expressing in silico enhanced human cytochrome P4503A4 for the degradation of leftover drug residues of amiodarone in vitro and in vivo. METHODOLOGY: In this study, cytochrome P4503A4 (1W0E) was taken as a template and the predicted 3D model of mutant CYP3A4 was developed using different bioinformatics tools. Selected mutant (Glu165Asp) protein was reverse translated and transcribed into cDNA sequence. The cDNA of CYP3A4 was synthesized, cloned into p427TEF construct and transformed into Saccharomyces cerevisiae OBS2. The degradation of leftover drug residues of amiodarone in vitro and in vivo using recombinant Saccharomyces cerevisiae OBS2 expressing CYP3A4 was evaluated. RESULT: The CYP3A4 activity in recombinant probiotic yeast was observed as 108 IU/mL and in vitro degradation of leftover drug residues of amiodarone was observed as 66.32 %. Whereas, in vivo degradation of leftover drug residues of amiodarone was observed as 72.61 % along with recovery of organ damage in histopathological studies of the animal model. CONCLUSION: Saccharomyces cerevisiae OBS2 expressing CYP3A4 can be used for probiotic and therapeutic applications.

Antimicrobial and anticancer potential of low molecular weight polypeptides extracted and characterized from leaves of Azadirachta indica.

Low molecular weight antimicrobial polypeptides were extracted and purified from the young fresh leaves of Azadirachta indica (neem). The total protein extracted was precipitated with 15% TCA-Acetone. The total purified proteins yielded from the two extraction methods were 122.33±2.21 and 115.09±1.88mg/g of the total fresh weight. The SDS-PAGE analysis identified the presence of eight low molecular weight polypeptide bands. The antimicrobial activity of the resolved bands was detected by Polyacrylamide gel-Agar overlay diffusion assay (PAG-ADA). Their broad-spectrum bactericidal activity was confirmed using the same technique and found three low molecular weight bands from 11 to 14kDa collectively exhibiting superior bactericidal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermis, Enterococcus faecalis, Pseudomonas aeruginosa and fungicidal activity against Candida tropicalis. The FTIR spectrum of the protein bands depicted the presence of hydroxyl and carbonyl groups in the protein bands. These polypeptides were characterized by MALDI-TOF/TOF analysis. Further, the purified protein extract was found to be active against HELA, BT-549 and Neuro-2a cell lines with IC50 value of 74.03±2.31, 64.82±1.64, 238.32±2.12 and 109.94±2.96, 59.61±0.75 for 24h and 48h, respectively. The results of present study indicate that these polypeptides exhibit broad spectrum antimicrobial and anticancer activity and can therefore be explored for their therapeutic potential.

Improved physicochemical pretreatment and enzymatic hydrolysis of rice straw for bioethanol production by yeast fermentation.

Lignocellulosic biomass such as agricultural and forest residues are considered as an alternative, inexpensive, renewable, and abundant source for fuel ethanol production. In the present study, three different pretreatment methods for rice straw were carried out to investigate the maximum lignin removal for subsequent bioethanol fermentation. The chemical pretreatments of rice straw were optimized under different pretreatment severity conditions in the range of 1.79-2.26. Steam explosion of rice straw at 170 °C for 10 min, sequentially treated with 2% (w/v) KOH (SEKOH) in autoclave at 121 °C for 30 min, resulted in 85 ± 2% delignification with minimum sugar loss. Combined pretreatment of steam explosion and KOH at severity factor (SF 3.10) showed improved cellulose fraction of biomass. Furthermore, enzymatic hydrolysis at 30 FPU/g enzyme loading resulted in 664.0 ± 5.39 mg/g sugar yield with 82.60 ± 1.7% saccharification efficiency. Consequently, the hydrolysate of SEKOH with 58.70 ± 1.52 g/L sugars when fermented with Saccharomyces cerevisiae OBC14 showed 26.12 ± 1.24 g/L ethanol, 0.44 g/g ethanol yield with 87.03 ± 1.6% fermentation efficiency.

Plausible exploitation of Jatropha de-oiled seed cake for lipase and phytase production and simultaneous detoxification by Candida parapsilosis isolated from poultry garbage.

Jatropha de-oiled seed cake was explored to utilize as a basic nutrient source for Candida parapsilosis, isolated from poultry garbage and selected based on the production of lipase and phytase enzymes under submerged fermentation. At optimized parameters under solid-state fermentation, lipase and phytase activities were recorded as 1056.66±2.92 and 833±2.5U/g of substrate (U/g), respectively. Besides enzyme production, complete elimination of phorbol esters and significant phytate reduction from 6.51±0.01 to 0.43±0.01g/100g of seed cake were noted after 3days incubation. Curcin and trypsin inhibition activity were reduced significantly from 26.33±0.43 to 0.56±0.02mg/100g and 229.33±2.02 to 11.66±0.28U/g, respectively after 5days incubation. Saponins were reduced from 5.56±0.19 to 1.95±0.01g/100g of seed cake after 7days incubation.

An analysis of horseradish peroxidase enzyme for effluent treatment.

The present study explains computational methods to design thermostable horseradish peroxidase enzyme using the crystal structure available from Protein Data Bank (PDB ID: 6ATJ). Multiple mutations were introduced to the original enzyme and developed a model by using Modeler9.14. After designing the model functional effect was confirmed in terms of protein ligand binding by molecular docking using Autodock 4.2. The implementation of modeling steps is demonstrated in the context of performing mutations for particular amino acid residue on the ligand pocket of the horseradish peroxidase, to derive the desired ligand binding properties. The docking investigation of modelled HRP with Quercetindihydroxide using Autodock 4.2 software that six amino acid residues, P139, H42, A31, L174, A38, and G169 are involved in hydrogen bonding. More importantly, it provides insight into understanding and properly interpreting the data produced by these methods. The 3D model was docked with Quercetindihydroxide (a known horseradish modulator) to understand molecular interactions at the active site region.